Axonal pathology represents an early, critical pre-symptomatic event in the disease course of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder that primarily targets motor neurons. The mechanism(s) by which axons degenerate in ALS are largely unknown. Collaborative efforts by the PIs and from others showed that pathogenic forms of the superoxide dismutase (SOD1) protein associated with familial forms of ALS (fALS) inhibit fast axonal transport (FAT). The potential disease relevance of these findings was highlighted by the discovery of fALS-related mutant genes encoding motor proteins, suggesting that FAT deficits suffice to cause axonopathy and degeneration of motor neurons. Our published and preliminary data demonstrated that the toxic effect of mutant SOD1 and misfolded forms of SOD1 on FAT was mediated through aberrant activation of a mitogen-activated protein kinase (MAPK) pathway. Importantly, our work established p38alpha (p38?), the major p38 MAPK isoform expressed in the CNS, as a specific MAPK component responsible for FAT inhibition and the motor protein kinesin-1 as a novel p38? substrate relevant to this toxic effect. Although multiple independent studies have demonstrated enhanced phosphorylation (and hence activation) of p38 MAPKs in ALS mouse models and in post-mortem human CNS tissues, none have addressed the contribution(s) of specific p38 MAPKs isoforms to the progressive degeneration on MNs triggered by mutant SOD1 in vivo. While pleiotropic therapeutic compounds that only mildly inhibit p38 MAPKs still showed a beneficial effect on the survival of fALS-SOD1 mice, findings from our work led us to posit that the therapeutic effects of p38 MAPK inhibition will be more substantial when the p38? isoform is specifically targeted. To define the contribution of aberrant p38? signaling to ALS pathogenesis, two PIs with a long history of collaboration and strong track-records in both ALS and aberrant kinase signaling have assembled a synergistic MPI application that will shed light on this important issue. Taking advantage of the SOD1G93A mouse model of ALS and of p38?AF/+ knock-in mice, which feature a mutant MAPK14 allele encoding non-activatable p38?, we will use genetic approaches to directly measure the contribution of aberrant p38? signaling to mutant SOD1-mediated motor neuron degeneration in vivo (Aim 1). It is also important to define mechanism(s) by which aberrant p38? activation causes axonal degeneration. To identify p38?-dependent alterations in the phosphorylation of axonal proteins induced by mutant SOD1, an unbiased phosphoproteomics approach will be applied to both the isolated squid axoplasm preparation, a unique and powerful ex vivo system with which to study axonal-specific events, and to cultured neurons prepared from SOD1G93A mice with attenuated p38? signaling (Aim 2). The outcomes of this Aim will move the field forward by providing novel mechanistic insights linking aberrant p38? activation to axonopathy and by revealing novel biomarkers to evaluate axonopathy in ALS and related neurodegenerative disorders. Collectively, this proposal aims to gain novel mechanistic insights into the pathogenic processes underlying ALS, and to establish in vivo contribution(s) of the specific molecular target p38? to the degeneration of motor neurons triggered by mutant SOD1.
The proposed research is directly relevant to public health because it addresses key molecular pathogenic pathways by which the disease-related SOD1 protein triggers degeneration of motor neurons and the onset of Amyotrophic Lateral Sclerosis (ALS), a devastating human neurodegenerative disease causing paralysis and eventual death of affected patients. The main goal of this proposal is aligned with the NINDS agenda because it evaluates mechanisms linking a specific, druggable molecular target (p38a) to motor neuron degeneration triggered by mutations in the SOD1 gene. This knowledge is expected to fully serve NINDS mission by devising proper therapeutic intervention strategies to reduce the burden of ALS and, potentially, other human neurodegenerative diseases.
